This invention is directed to a serum-free and mitogen-free growth factor for thymus-derived lumphocyte (i.e., T-cell growth factor) and a process of producing same. This invention is also directed to the use of the above-mentioned T-cell growth factor in the treatment of patients suffering from tumors and in improving the growth of natural killer cells in such patients.
T-cell growth factor (TCGF), also referred to as Interleukin 2 (IL 2) is a protein which functions to activate blastogenesis and the proliferation of cytotoxic lymphocytes dependent on the thymus (T-cells). Hereinafter, T-cell growth factor (TCGF) and Interleukin 2 (IL 2) will be used interchangeably to designate the same material. IL 2 is defined by its ability to support growth of T-lymphocyte lines in cell cultures. IL 2 activates a subpopulation of T-cells which is distinguished by specific cytotoxic properties and natural killer properties. "Natural killing" refers to the ability of such cells to kill tumor cells without previous sensitizing. These cells represent the most important effector cells in the immune reactions against spontaneous malignant tumors. Natural killer cells are important since they represent the first line of defense in the body against tumors. It has been found that the activity of natural killer cells in tumor patients is suppressed. Therefore, when the natural killer cells in the patient are stimulated into activity, the growth of the tumor cells can be stopped.
In addition, it has been suggested that IL 2 may be used in the defense against viral deseases. Furthermore, a decisive role has been attributed to IL 2 in the rejection of transplants. By suppressing the production of IL 2 in the patient after a transplantation operation, the probability of the patient rejecting the transplant is substantially decreased.
As mentioned above, tumor patients have been found to be defective in the function of the cellular immune system attributable to a defect in the in vitro ability to produce IL 2. If it is assumed that the tumor causes and must cause this defect in order to survive, a therapeutic procedure can be established by supplying the patient with preparations containing IL 2 in order to activate the inactive natural killer cells.
Such a therapeutic procedure is similar to that for interferon. Recently, most interest has been directed to the effect of interferon on cancer. Interferon is known to be effective in killing virus-infected cells, although its effect on cancer is still unknown. It is of interest to note that it is species specific, i.e., interferon, to be effective on humans, must be extracted from human blood. On the other hand, IL 2 produced with cells from bovine blood and spleen has been found to activate human cells. This also applies to porcine blood and spleen. However, IL 2 extracted from mice or rats will not activate human cells since the donor belongs to a species which is too biologically unrelated to humans. IL 2 extracted from large animals such as cows, pigs and sheep will activate human cells. This is significant in that with IL 2, the risk of introducing into the patient hepatitis virus carried in the donor's blood can be avoided by using bovine (cattle) or porcine (pig) lymphocytes. Another advantage is that the supply of bovine and porcine lymphocytes is much greater than that of human cells.
According to Lucas, et al., "Parameters of Lymphocyte Activation," IL 2 is not identical to interferon but occurs in stimulated leucocyte supernatants containing interferon. Possibly, part of the therapeutic effect of leucocyte interferon preparations is due to the IL 2 contained therein.
The production of IL 2 has been discussed by Alvarez, et al., "Human T-Cell Growth Factor," J. of Immunology, 123, 3, 977 (1979) and Gillis, et al., "T-cell growth factor: parameters of production and quantitative microassay for activity," J. of Immunology, 120, 2027 (1978). In general, IL 2 is produced by stimulating peripheral mononuclear blood cells with a mitogen or antigen.
The production of a phytohemagglutinin-(PHA)-free T-cell growth factor is discussed in Fagani, et al., "Removal of PHA from conditioned medium by affinity chromatography," J. of Immunological Methods, 33, 313 (1980). The procedure described therein is not only cumbersome but also provides a product which contains serum. The presence of serum which contains numerous proteins may mask the true effect of IL 2 and repeated injections of such a serum-containing preparation regularly cause allergic and anaphylactic reactions in patients. Furthermore, a serum-containing IL 2 cannot be concentrated since the serum will precipitate and plug the pores in the filter.
The present inventors have successfully produced serum-free and mitogen-free T-cell growth factor. Heretofore, it has been accepted that serum is necessary for cell growth. As a result, serum is always used in cell culture. Through serum-free production, the present growth factor can be highly concentrated and the activity thereof can be increased between 300 and 500 times. Furthermore, since no toxic substances, such as lectins, for example, are present in the growth factor, the cells from which the growth factor is extracted can be used several times before losing its activity. As a result, the efficiency of the extraction process is substantially improved over the prior art. Thus, the present invention is believed to provide a clear and significant advance in the art.